Focal segmental glomerulosclerosis (FSGS) is an intractable glomerular disease with few treatment options and poorly understood etiology. Activating mutations in TRPC6 can cause FSGS7,8 and disease-associated TRPC6 mutant forms can cause NFAT-dependent transcription in vitro9. We have generated a murine model in which inducible podocyte-specific NFATc1 activation leads to FSGS, providing the first in vivo proof of the involvement of NFAT signaling in FSGS. We have also found that NFAT-activation in podocytes specifically upregulates Wnt6 and Fzd9. We hypothesize that NFAT signaling occupies a key position in the regulatory network in podocytes such that its misregulation alters signaling pathways, such as the Wnt pathway, crucial for the homeostasis and function of podocytes, leading to FSGS. To test this hypothesis and to further delineate the pathogenic mechanism of FSGS, we will first determine if canonical Wnt signaling is essential for the effects of NFAT activation in podocytes (Aim 1). We will then delineate the pathogenic mechanism by which NFAT activation in podocytes leads to FSGS, focusing on the reversibility of the disease, the potential association between NFAT activation and human FSGS, as well as the involvement of Nephrin and actin cytoskeleton alterations (Aim 2). Finally, we will use the latest genome technologies to reveal transcriptional alterations and additional NFAT targets in association with the development of podocyte dysfunction and FSGS (Aim 3). Although the anti-proteinuric effects of cyclosporine (a calcineurin inhibitor) have been attributed to the inhibition of dephosphorylation of synaptopodin by calcineurin10, suppression of NFAT signaling downstream of calcineurin may also play a role. Thus, in addition to defining the mechanism by which NFAT/Wnt pathways operate in podocytes and in the pathogenesis of FSGS associated with TRPC6 mutations, this study will also contribute to better understanding of the anti- proteinuric effects of calcineurin inhibitors. The unbiased genome-wide analyses will go beyond the analysis of known factors associated with FSGS to potentially reveal previously unsuspected factors in the pathogenesis of FSGS. Results from these studies may form the theoretical foundation for treatments of FSGS targeting NFAT or factors downstream of NFAT that may be more specific and with fewer side-effects than those aimed at more upstream factors, such as calcineurin and TRPC6. PUBLIC HEALTH RELEVANCE: Focal Segmental Glomerulosclerosis (FSGS) is a type of intractable glomerular disease with devastating outcomes, few treatment options, and poorly understood etiology. The proposed studies will delineate the mechanism by which NFAT and Wnt signaling pathways operate in podocytes and will contribute to better understanding of the pathogenesis of FSGS associated with TRPC6 mutations and the anti-proteinuric effects of calcineurin inhibitors. In addition, the unbiased genome-wide analyses will go beyond the analysis of known factors associated with FSGS to reveal previously unsuspected factors in the pathogenesis of FSGS. Results from these studies may form the theoretical foundation for treatments of FSGS targeting NFAT or factors downstream of NFAT that may be more specific and with fewer side-effects than those aimed at more upstream factors, such as calcineurin and TRPC6.